The distribution of cells within organs, their involvement in tissue structure, and their organization in three-dimensional networks are important aspects in the analysis and understanding of biological processes. Here, we present a protocol for preparing whole-mount urethra and urinary bladder from transcardially perfused mice for immunolabeling and analysis by confocal laser scanning microscopy. We describe steps for perfusion, preparation, tissue clearing, and analysis using confocal laser scanning microscopy. We then detail procedures for three-dimensional processing of the murine urethra and urinary bladder.For complete details on the use and execution of this protocol, please refer to Schmidt et al.1

Cholinergic urethral tuft cells (UTCs) are regarded as sentinels of microbial products in the urogenital tract, initiating reflex micturition as a protective mechanism. We here hypothesize that acetylcholine released by stimulated UTC leads to neurogenic inflammation by triggering neuropeptide (substance P and calcitonin gene-related peptide) release from nearby sensory nerve terminals. In the mouse urethra, we find that peptidergic nerve fibers expressing a nicotinic acetylcholine receptor are in contact with UTC. Optogenetic activation of UTC and the UTC activator denatonium leads to the release of neuropeptides from explanted urethrae through cholinergic nicotinic signaling. In vivo, intraurethral application of denatonium induces plasma extravasation, a hallmark of neurogenic inflammation, which is sensitive to genetic interruption of the UTC intracellular signaling cascade (Trpm5-/-) and blockade of the substance P receptor: neurokinin-1 receptor. Thus, UTCs not only trigger long-distance reflexes involving the bladder but also evoke neurogenic inflammation, representing a local defense reaction.

Increased sugar concentrations on mucosal surfaces display risk factors for infections. This study aims to clarify sugar monitoring in the urethra. Urethral tuft cells (UTC) are known sentinels monitoring the urethral lumen for potentially harmful substances and initiating protective mechanisms. Next-generation sequencing (NGS), RT-PCR, and immunohistochemistry show expression of the taste receptor Tas1R3 in murine UTC, a crucial component of the classical sweet detection pathway. Isolated UTC respond to various sugars with an increase of intracellular [Ca2+]. The Tas1R3 inhibitor gurmarin and Tas1R3 deletion reduces these responses. Utilizing mice lacking UTC, glibenclamide, a K+-ATP channel antagonist, and phlorizin, a SGLT1 inhibitor, reveal an additional Tas1R3 independent sweet detection pathway. Inhibition of both pathways abrogates the sugar responses. Rat cystometry shows that intraurethral application of sucrose and glucose increases detrusor muscle activity Tas1R3 dependently. Sugar monitoring in the urethra occurs via two distinct pathways. A Tas1R3 dependent pathway, exclusive to UTC, and a Tas1R3 independent sweet detection pathway, which can be found both in UTC and in other urethral epithelial cells.

Urinary Tract Infection Increased sugar concentrations display risk factors for infections. Article number 2400117 by Klaus Deckmann and co-workers clarifies sugar monitoring in the urethra. Urethral tuft cells (UTC) are sentinels monitoring the urethral lumen and initiating protective mechanisms. Monitoring occurs via two pathways. A UTC/Tas1R3 dependent and a Tas1R3 independent pathway, found in both UTC and in other urethral epithelial cells. Sugars increases detrusor muscle activity UTC/Tas1R3 dependently.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

We have recently identified a cholinergic chemosensory cell in the urethral epithelium, urethral brush cell (UBC), that, upon stimulation with bitter or bacterial substances, initiates a reflex detrusor activation. Here, we elucidated cholinergic mechanisms that modulate UBC responsiveness. We analyzed muscarinic acetylcholine receptor (M1-5 mAChR) expression by using RT-PCR in UBCs, recorded [Ca2+](i) responses to a bitter stimulus in isolated UBCs of wild-type and mAChR-deficient mice, and performed cystometry in all involved strains. The bitter response of UBCs was enhanced by global cholinergic and selective M2 inhibition, diminished by positive allosteric modulation of M5, and unaffected by Ml, M3, and M4 mAChR inhibitors. This effect was not observed in M2 and M5 mAChR-deficient mice. In cystometry, M5 mAChR-deficient mice demonstrated signs of detrusor overactivity. In conclusion, M2 and M5 mAChRs attenuate the bitter response of UBC via a cholinergic negative autocrine feedback mechanism. Cystometry suggests that dysfunction, particularly of the M5 receptor, may lead to such symptoms as bladder overactivity.

Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl- baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.47 μM). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing α-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of α-ENaC in cholinergic UBC-salt perception, homeostatic ion transport, mechanoreception-remains to be determined.

Purpose of review A specialized epithelial cell with chemosensory properties of taste cells known from the mouth has been newly identified in the urethra and linked to pathogen recognition. We here describe its properties and its link to defence mechanisms, showing parallels to similar sentinel cells in the respiratory and gastrointestinal tract. Recent findings In the urethra, slender epithelial cells with apical microvilli (‘brush cells’) express bitter and umami taste receptors and the downstream signalling cascade known from oropharyngeal gustation, utilizing it to monitor for bacterial products and bacterial growth facilitating conditions. Upon stimulation, they release acetylcholine, and their sensitivity is subjected to cholinergic feedback. They are approached by cholinoceptive sensory nerve fibres, and intraurethral bitter application evokes reflex detrusor activity. Similar cells in the respiratory and gastrointestinal mucosa additionally regulate immune function through local neurogenic inflammation and cytokine release, triggered by bacterial products and parasites. Summary This cell is interpreted to serve as chemosensory sentinel for potential hazardous compounds in the urethral lumen, triggering a protective mechanism (flushing through micturition) against further ascent. Dysfunction may be related to higher risk of infection or inadequate detrusor activity, pharmacological intervention may be considered to combat infection or detrusor overactivity.

Airway function is profoundly controlled by parasympathetic, sympathetic and peptidergic sensory nerve fibers. Traditionally, the function of airway innervation is studied by electric field stimulation or by pharmacologic activation or inhibition. These methods allow only poor discrimination between fiber subtypes. Optogenetics is a new promising tool to solve these questions of specificity and selectivity. A blue light sensitive channelrhodopsin 2 (ChR2) from Chlamydomonas has been inserted in mice to create cell-specific expression of ChR2. Light stimulation induces depolarization and release of neurotransmitters from ChR2-expressing neurons. We here generated two mice strains expressing ChR2 in the major subtypes of neurons expected to induce bronchoconstriction, i.e. cholinergic (parasympathetic, expressing choline acetyltransferase = ChAT) and peptidergic sensory neurons, expressing transient receptor potential cation channel subfamily V member 1 (TRPV1), respectively, by crossbreeding ChAT and TRPV1 cre driver lines with Ai27D mice, expressing a ChR2-tdTomato fusion protein following exposure to Cre.

A peculiar cell type of the respiratory and gastrointestinal epithelia, originally termed “brush cell” or “tuft cell” by electron microscopists because of its apical tuft of microvilli, utilizes the canonical bitter taste transduction cascade known from oropharyngeal taste buds to detect potential hazardous compounds, e.g. bacterial products. Upon stimulation, this cell initiates protective reflexes and local inflammatory responses through release of acetylcholine and chemokines. Guided by the understanding of these cells as sentinels, they have been newly discovered at previously unrecognized anatomical locations, including the urethra. Solitary cholinergic urethral cells express canonical taste receptors and are polymodal chemosensors for certain bitter substances, glutamate (umami) and uropathogenic Escherichia coli. Intraurethral bitter stimulation triggers cholinergic reflex activation of bladder detrusor activity, which is interpreted as cleaning flushing of the urethra. The currently known scenario suggests the presence of at least two more urethral chemosensory cell types: non-cholinergic brush cells and neuroendocrine serotonergic cells. The potential implications are enormous and far reaching, as these cells might be involved in monitoring and preventing ascending urinary tract infection and triggering of inappropriate detrusor activity. However, although appealing, this is still highly speculative, since the actual number of distinct chemosensory cell types needs to be finally clarified, as well as their embryological origin, developmental dynamics, receptor equipment, modes of signalling to adjacent nerve fibres and other cells, repertoire of chemo- and cytokines, involvement in pathogenesis of diseases and many other aspects.